CN102504027A - Preparation of multi-epitope thymidine kinase 1 (TK1) antibody and use of multi-epitope TK1 antibody for early tumor detection and risk early warning in mass physical examination screening - Google Patents
Preparation of multi-epitope thymidine kinase 1 (TK1) antibody and use of multi-epitope TK1 antibody for early tumor detection and risk early warning in mass physical examination screening Download PDFInfo
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Abstract
The invention provides a high-specificity and high-sensitivity coordinated antibody against human thymidine kinase 1 (TK1) prepared from an antigenic determinant consisting of 23 peptides at an N terminal, 20 peptides at a C terminal and 28 peptides at the C terminal of the TK1 monomer from a human hela cell and the use of the detection and diagnosis system of the antibody in tumor diagnosis. The antigenic determinant comprises the following amino acid sequences: the sequence of the 23 peptides (3-25) at the N terminal:CINLPTVLPGSPSKTRGQIQVIL; the sequence of the 20 peptides(206-225) at the C terminal: CPVPGKPGEAVAARKLFAPQ; and the sequence of the 28 peptides (198-225) at the C terminal: AGPDNKENCPVPGKPGEAVAARKLFAPQ. The invention also provides a method for preparing the antibody by using the antigen. An antibody kit provided by the invention has the characteristics of high sensitivity, high specificity, low cost and the like; and the early tumor can be detected and pre-warned in mass physical examination screening by enhanced chemiluminescence dot blot assay, immuno-histochemistry and the detection kit.
Description
Technical field:
The present invention relates to a kind of antibody and application thereof, specifically relate to the preparation of a kind of high specific, highly sensitive multi-epitope TK1 antibody and in crowd's health check-up examination infantile tumour detect and Risk-warning in application.
Background technology:
Cancer is human dead killer; Although many image detection methods and treatment means are arranged, for example: chemotherapy, endocrine therapy, radiotherapy and operation etc., though very big progress has been arranged; But possibly reach perfect degree far from, with the possibility of recurrence and transfer.Therefore, early stage discovery, particularly discovery and the early stage treatment early than the precancerous lesion phase is the best Gospel that gives patient's rehabilitation.
Cancer is a kind of chronic disease of cellular abnormality propagation.Cancer cells propagation unusual with out of control is that though abnormal gene expression, therefore not canceration as yet is called as the preceding disease of precancerous condition or cancer owing to certain has taken place or various mutations is caused with the relevant gene of propagation in the normal cell.Disease is meant that the people who suffers from these diseases will increase than the chance that other people get cancer greatly before the cancer, thereby also is called as preceding disease of cancer or precancerous condition.And according to the cytopathology notion, one type of proliferative lesion with cell atypism and prosoplasia is called precancerous lesion.Cell carcinogenesis is phase in and develops gradually.Attack normal tissue cell to producing cancer from carcinogen, generally need more than 10 years even the longer time, advance rate and course of disease length depend primarily on tumor type and individual difference.Disease progression becomes in this very long process of Cancerous disease before the cancer, also be divided into gently, in, heavily wait different steps.The severe precancerous lesion be before malignant tumour takes place last, also be the most dangerous period.Obviously, preventing and treating precancerous lesion is that prophylaxis of tumours and tumour are cured the most key link, by being paid close attention in the whole world.Yet time and position that precancerous lesion takes place all have uncertainty, and pathology still is in cell levels, not different from sings and symptoms, and existing detection means is difficult to inspection discovery in time.And conventional blood test (formed elements in the blood is that the quality and quantity of these three systems of red corpuscle, white corpuscle and thrombocyte detects and analyzes) and the urine detection used in the existing health check-up can provide the information of abnormal index such as serum or urine protein or enzyme and relative disease; (B-is ultra for existing conventional physics image detection method; X-light); The polyp or the polyp that can be observed in the histoorgan increase, the relevant information of tumour or suspected tumor, but these methods still can not be known the polyp that shows in examinee's histoorgan; The abnormality proliferation situation of the tumour cell of tumour or lump, whether very difficult assessment might process be malignant tumour.
What particularly point out is, but up to the present, seldom has believable tumor growth correlating markings thing to be applied to the health check-up examination.2008, diagnosis and supervision that how U.S. Cigna health care company will be at present be used for cancer with tumor markers (Tumor Markers) were collected, and proposed the criterion and the practice guideline of generally acknowledging, for the blood serum designated object that tumour is correlated with, assessed.Carry out retrospective research for existing many tumor markerses; CEA for example, CA15-3, a series of marks such as CA199 and AFP; Because this type mark is not directly related with the propagation of tumour cell; They normally can not detect with the level of independent a kind of tumor markers and the diagnosing cancer disease, and this type code thing raises, and possibly indicate has cancer; But it is cancer that their existence can not be diagnosed, and these marker detection are to need to combine other diagnostic modes (for example: biopsy, radiation/imaging) just can confirm as cancer.
Later 1950s, the investigator finds thymidine kinase (EC.2.7.1.21 is called for short TK for TK, ATP:thymidine5 '-phosphotransferase), a kind of enzyme of pyrimidine salvage pathway, and catalysis thymidine phosphoric acid turns to thymidylic acid.TK occurs with the form of two kinds of isozyme in people's cell: cytoplasmic thymidine kinase (being called for short TK1) and thymidine kinase,mitochondrial (being called for short TK2).Thymidine kinase 1 (is called for short TK1; Be also referred to as cytoplasmic thymidine kinase) be and class mark of closely-related cell cycle of cell cycle regulating; A large amount of fundamental researchs subsequently and experimental oncology have proved that the TK1 expression is closely connected with cell growth state; Therefore TK1 can be used as the proliferation activity that a kind of good cell proliferation affinity tag detects proliferative cell, and the propagation degree that therefore will be suitable for malignant tumour detects.
Adopt people TK1 antibody test serum T K1 concentration level more can truly represent the cell that whether has abnormality proliferation in the collective, therefore, in nearly more than 10 years, according to the Antibody Preparation principle, a kind of TK1 antibody is tried out in clinical practice.A plurality of team have prepared evaluation and the monitoring that different TK1 antibody is used for tumor disease, but are not suitable for conventional crowd's health check-up examination.
On this basis; We are further screening study in 2002; This crucial immune sequence-31 peptide (195-225) of having selected to be exposed to the antigenic determinant C end parts of TK1 protein surface has prepared anti-people TK1-IgY antibody (Chinese invention patent WO204 100760A1, international patent W O 204100760A1.25/11-2004).Employing people TK1C holds the anti-people TK1-IgY antibody of 31 peptides preparation and utilizes the seroimmunity and the histogenic immunity lesion detection test kit of its preparation; This antibody will detect activated TK1 albumen and inactive TK1 albumen simultaneously, also can detect the mixture that TK1 protein and other molecule form.Although this serum reagent box-enhancing electrochemiluminescent immunoassay dot blot detection system is tried out in the health check-up examination, sensing range is between 0.3-2pM, when 0.1-1pM; Error adopts crowd routine physical examination serum T K1 and cancer patients's serum T K1 to carry out the analysis of ROC value sometimes greater than 30%, and ROC value TG-AUC is 0.96; Specificity is 0.991, and maximum likelihood method ratio (+) is 56.04, proves that the specificity of antibody is fine; Can be used for crowd's health check-up examination; But sensitivity is 0.51%, and the antibody that needs further to prepare detection sensitivity height, specificity and good stability improves detection system, so that better improve the detection tolerance range in the 0.1-1pM scope.
Summary of the invention:
Adopt on the TK1 monomer of human cervical carcinoma cell C hold 31 peptides (195-225) as antigenic basis on; Through practice for many years; Filtered out the multiple spot position special antigen determining family that is exposed to the TK1 protein surface, comprised key immune peptide sequence 23 peptides of N end parts, C holds crucial immune sequence 28 peptides of 20 peptides and C end parts; Use the immune sequence of above-mentioned key and do special antigen, the anti-people TK1-IgY antibody that adopted final purifying of hen immunity and combined preparation.Compare with the commercial anti-people TK1-IgG antibody in the existing world; Prepared antibody has purity height (＞99%), output height, highly sensitive, high specific and does not have the non-specific immunity cross reaction; And can preserve more than 1 year and activity stabilized in+4-8 degree refrigerator, be convenient to advantages such as commercial transportation, preservation and operation.Anti-people TK1 multi-epitope antibody of the present invention and TK1 detection system; The propagation degree that can reflect cell really; Thereby can be under situation about detecting early than iconography, the early stage potential abnormal cell proliferation of early warning in the health check-up crowd, the caution examinee possibly process be the risk of malignant tumour.
Technical scheme provided by the invention comprises:
1, design and screening antigen
The peptide section of N, C end on the protein surface of selecting to expose.
2, preparation antibody
Utilize crosslinked composition 23 peptides of immune sequence of above-mentioned key and KLH-KLH, 20 peptides-KLH, the antigen of 28 peptides-KLH go immune hen to make respectively.And utilize the advantage of hen immunity to be: there is the molecular genetic otherness between IgY of (1) chicken and people's the IgG; (2) TK1 of chicken and people's TK1 has racial diversify; (3) compare with the immune polyclonal antibody for preparing of traditional rabbit, IgY has endogenous molecule homogeneity (only producing one type antibody molecule, i.e. IgY); (4) IgY antibody does not activate people's complement system, thereby partly blocks the activation of non-specific antigen binding site in the human serum; (5) Rheumatoid factors, polyclonal (RF) not with the IgY antibody response.This RF is the main source of non-specific responding in many immunoassay, because the Fc partial reaction of RF and Mammals IgG antibody, can cause the false positive of patient and healthy subjects serum; (6) present most of patient with breast cancer needs assisting therapy, and many patients accept chemotherapy; For example: He Sai is left alone without help, targeted therapy medicine, monoclonal antibody; Yet need the hormone antagonist treatment later on, particularly under the Antybody therapy situation, TK1-IgY antibody demonstrates than monoclonal antibody health check-up and surveys the advantage that has more.
3, antibody purification
Utilize said N to hold 23 peptides, C holds 20 peptides, and C holds 28 peptide sequences crosslinked with KLH respectively, and immune hen extracts yolk liquid respectively, adopts water extraction preparation antibody-solutions, and the affinity column with the preparation of the antigenic determinant among the present invention comes antibody purification respectively.
Said purifying comprises the steps:
The first step; Just sift out the antibody that immune hen produces and whether have high specific and highly sensitive; Promptly the yolk liquid to each immune hen separates, and obtains the antibody bullion after the extraction of employing water, obtains the high specific and the highly sensitive anti-people TK1 antibody of primary dcreening operation through affinitive layer purification.Type according to immunizing antigen is selected affinity column, and mixed bullion liquid is to derive from 23 peptides-KLH, 20 peptides-KLH, and the anti-TK1-IgY antibody of the antigen immune of 28 peptides-KLH, the affinity column preparation is necessary for N and holds 23 peptides, and C holds 20 peptides and C to hold the affinity column of 28 peptides.
Second step, the affinity column of preparation combination immunizing antigen, the proportioning test to 3 kinds of antibody that just sift out make up is characterized in that the selection of combined antigen affinity column proportioning.The preparation affinity column requires the blending ratio according to selected 3 kinds of antibody, calculates corresponding antigenic determinant, i.e. 23 peptides, the per-cent of 20 peptides and 28 peptide amounts.After the test of affinity column chromatography purifying, select best proportioning assembled scheme.
4, the anti-people TK1-IgY of multidigit point antibody combined preparation antibody, unless otherwise indicated, the per-cent among the application is mass percent.
Ratio range: the monospecific antibody of 23 peptides immunity accounts for 25%～45%, and the monospecific antibody of 20 peptides immunity accounts for 20%～40%, and the monospecific antibody of 28 peptides immunity accounts for 20%～55%.
5, identify the sensitivity and the specificity of anti-people TK1-IgY antibody
1) confirm that whether preparation antibody only have specific reaction with people's TK1, the first step adopt TK1 positive with the negative cells strain, the western method of printing and dyeing is identified the specificity of antibody: utilize lymphoma positive strain CEM TK1
+With negative strain CEM TK1
-Identify.Behind the natural gum electrophoresis, carry out the western immunity printing and dyeing of TK1 antibody, CEM TK1 is positive, and strain has a tangible TK1 electrophoresis band, and the negative strain of CEM TK1 does not manifest any band.What confirm antibody is only with the TK1 enzyme immunoreation to be arranged, and is specific antibody.
2) whether the antibody of confirming preparation only have specific reaction with the serum T K1 of tumour patient; Second step adopted and selects to have confirmed as healthy no disease patient's the serum and the serum specimen of the positive value of tumour patient; Detect with strengthening electrochemiluminescent immunoassay dot blotting method; The serum specimen of the positive value of tumour patient is answered the positive value of showed different, and healthy person should be lower than detection threshold 2pM or not show immunoreation.The serum of having confirmed antibody and healthy no disease patient does not have immunological cross-reaction, is specific antibody, and the intensity of STK1 is reacted among the comparison of tumor patients serum, and the different antibodies of just sifting out is identified, filters out highly sensitive and the good antibody of specificity.
3) confirm preparation antibody whether only with tumour patient in TK1 specific reaction is arranged; From 2) filter out highly sensitive and the good antibody of specificity the step; Adopt first visit but do not treat the serum of tumour patient and healthy no disease patient's serum specimen point sample, carry out the natural gum electrophoresis after, again with western immune printing and dyeing of anti-TK1 antibody; The serum specimen of tumour patient shows a tangible TK1 electrophoresis band, and healthy no disease patient's serum specimen does not manifest any band.Confirming that antibody is only with the serum T K1 enzyme of tumour patient immunoreation to be arranged, is specific antibody.
The present invention utilizes aforesaid combination antibody under 100,000 grades clean environment, and antibody is carried out packing, assembles the TK1 immunity detection reagent, and combines with chemiluminescent analyzer, is applied to infantile tumour detection and Risk-warning in crowd's health check-up examination.
This invention relates to early prediction; Can detect early than iconography under the situation of cancer; Be used to detect the preceding disease of the polytype cancer/tumor disease of hiding in early days/chronic disease and abnormal cell proliferation relevant with tumor disease, the early warning examinee possibly process be the risk of malignant tumour.STK1 detect to get into the health check-up set meal, the risk population that screening STK1 raises, and the disease joint assessment cells in vivo abnormality proliferation situation relevant with following malignant tumour process comprises: regularly the dynamic observing of precancerosis disease risk population; In for example organizing/and the severe hyperplasia, the polyp pathology, in/severe ulcer or erosion, viral hepatitis is positive, and for example hepatitis B 135/145, refractory type anaemia disease or the like; The regular monitoring relative disease possibly fade to cancer disease risks process, innocent tumour for example, in/severe fatty liver, overweight, long-term chronic inflammatory diseases relevant or the like with tumor disease; Tumor recovering patient crowd regular monitoring, Risk-warning; Have or not cellular abnormality propagation in the early warning body: 3-6 month STK1 continues high-level person; But existing Absence of physical signs disease. cellular abnormality propagation is arranged in the early warning body, need and coherent video appearance, tumor markers; Serum and urine index associating regular monitoring, the observation of cell abnormality proliferation changes.
MEC detects examinee STK1 level, sets up the basal level of the individual STK1 of healthy person, does not for example have the healthy examinee of tumor disease; Need get first sample in 1 month one after another, analyze the level of STK1,3 middle of the month; Carry out duplicate detection 3 times; 3 times detected result is calculated MV, draw the basal level of the individual STK1 of healthy person, the healthy examinee of no tumor disease generally is being lower than 1pM; During the later annual routine physical examination, compare with the basal level of the STK1 value that detects with the STK1 of individuality.
Possible process is that the examinee's of cancer disease risks prediction is the basic value and later annual test TK1 value according to the TK1 of individuality, to both STK1 level comparisons.For example; Examining the basis of STK1 value and individual STK1 now compares; If should confirm to have the preceding disease of cancer/innocent tumour disease/chronic disease relevant by individuality with tumor disease; But the level of TK1 still is lower than risk threshold value, and it is low that the examinee is evaluated as cellular abnormality propagation level, and process is that the risk of cancer disease is low; If should confirm to have the preceding disease of cancer/innocent tumour disease/chronic disease relevant with tumor disease by individuality, the level rising 1-2 of STK1 doubly surpasses 2pM, and the examinee is evaluated as cellular abnormality propagation level and raises, and the early warning process is that the risk of cancer disease raises.
Adopt this method that the examinee is regularly detected, can monitor the individual several years, so that many decades judges whether the examinee is in cancer disease low risk or high risk condition.Therefore, this invention gives examinee and MEC doctor's early stage information.If STK1 raises, the early warning examinee has the excessive risk of cancer disease process; Might advise the therapeutic intervention scheme of being correlated with giving the killer opportunity that the examinee keeps away from risk and healing to the doctor
With the prior art ratio, advantage of the present invention is following:
(1) the prepared antibody of the present invention has purity height (＞99%), output is high, high specific does not have the non-specific immunity cross reaction and accuracy of detection can reach 0.1pM.Experimenter's working curve is represented: ROC TG-AUC value=0.96 (p＜0.0001), it is 236.49 that maximum likelihood method ratio (+) obviously raises, and specificity is up to 0.997, and sensitivity is up to 0.737.
(2) be applicable to crowd's health check-up examination; Can be according to the variation of different individual instances of examinee and serum T K1 (STK1) level greater than 2pM; Indication abnormal cell proliferation degree provides information for individual and doctor, and strick precaution/therapeutic intervention reasonably keeps healthy; Keep away from risk, possibly greatly improve patient's curative effect.
(3) good stability can be preserved more than 1 year and activity stabilized the commercial transportation of being more convenient for and the implicit costs of preserving and can reduce reagent in+4-8 degree refrigerator.
Description of drawings:
Fig. 1: ABmart antibody analysis test TK1 protein graphical spectrum.
Fig. 2: (a) TK1 antibody kit point sample figure as a result
(b) STK1 dot blotting experimental analysis software test TK1 antibody kit point sample data
(c) STK1 dot blotting experimental analysis software test TK1 antibody kit point sample data and curves figure
Fig. 3: (a) be CEM TK1
+With CEM TK1
-Western part printing and dyeing result.
(b) for identifying lymphatic cancer patient before the treatment, before the treatment/after head and neck cancer patient, western part printing and dyeing result of healthy subjects serum.
Fig. 4: CEM TK1
+, CEM TK1
-, endocervix cancer and colorectal carcinoma TK1 antibody staining result
Fig. 5: the comparison diagram of vitamin H-TK1-IgY antibody (2 footwork) and three-step approach
Fig. 6: the ROC chart that the serum T K1 that MEC provides detects
Fig. 7: the anti-TK1-IgY antibody of Hua Ruitong health and the experiment comparison diagram of the anti-TK1-mAb antibody of commercially available commercialization
Embodiment:
With following embodiment the present invention is described in detail:
Embodiment 1: select the specificity multi-epitope antigen
As shown in Figure 1, with software prediction TK1 no signal peptide, do not have and stride membrane structure.Diagram strand TK1 protein 23 4 amino acid distribute, and point out amino acid whose hydrophilic and hydrophobic amino acid distribution uniform.Collection of illustrative plates provides possibility: 1) considers from the antigenicity angle, and better at the polypeptide that the high place of blue curve peak value is selected; 2) according to the distribution of hydrophilic amino acid, select hydrophilic region; 3) select the stronger die aromatischen Aminosaeuren of hydrophobicity shown in the negative value.
Instance 2: the antigen of selecting highly sensitive, the anti-people TK1-IgY antibody of high specific multidigit point combination
Select the antigenic selection of preparation highly sensitive, the anti-people TK1-IgY antibody of high specific multidigit point combination: have identical epi-position because the partial order of human cervical carcinoma TK1 is shown with other albumen; The antibody that obtains possibly have cross reaction with other nonspecific proteins; Purification difficult according to announcing known TK1 sequence (protein library), selects to be exposed to the special peptide of protein surface; Be listed in the table below greater than 85% polypeptide fragment according to the degrees of specificity of the polypeptide that obtains with software prediction; The antigen fragment of the TK1 high specificity of screening has designed TK1N and has held 23 peptides, and C holds 20 peptides and C to hold 28 peptides as haptin.
Embodiment 3: screening specificity multi-epitope antigen
The selection of specificity multi-epitope antigen: according to known TK1 space structure; Selection is exposed to the antigenic determinant on the proteinic surface of TK1; According to the strong antigen fragment of instance 2 screening TK1 immunitys, the N of screening TK1 holds 23 peptides, and C holds 20 peptides; Holding 28 peptides with C is the strong antigen fragments of immunity; Three sections aminoacid sequences (are seen sequence table) respectively as follows: 1) N holds 23 peptides (3-25): CINLPTVLPGSPSKTRGQIQVIL, 2) C holds 20 peptides (206-225): CPVPGKPGEAVAARKLFAPQ, 3) C holds 28 peptides (198-225): AGPDNKENCPVPGKPGEAVAARKLFAPQ.
Embodiment 4: make up anti-people TK1-IgY antibody with the coordination of hen immunity preparation specificity multi-epitope
Use above-mentioned N and hold 23 peptides, C holds 20 peptides, C hold 28 peptide sequences do haptin respectively with the KLH hinge, the preparation immunizing antigen, utilize the new combinatorial antibody of anti-people TK-IgY antibody that this antigen prepd is highly sensitive, specificity is good.The antigen consumption: select 40 of the good healthy Luo Man hens of immunity, consumption is 0.5 milligram, KLH N23 peptide; The KLH-C20 peptide; The KLH-C28 peptide mixed with the Fu Shi Freund's complete adjuvant above-mentioned immunizing antigen with the dissolving of PBS damping fluid again by 1: 1, be expelled to the chest muscle of bird inlay.Through twice to chicken with freund 's incomplete adjuvant and mixed antigen liquid booster immunization, every day is collected egg in the back all around, descends storage at 4 ℃.
Embodiment 5: extract and purifying multidigit point combinatorial antibody
The purifying crude process of antibody is following: the funnel that adopts the band mesh is with yolk and albumen sepn, and complete yolk is cleaned the back with vaal water and removed the yolk epidermis with taking the photograph son, collects egg yolk liquid.With the distilled water diluting egg yolk liquid of 10 times of volumes, mixing and stirring well even back adjust pH is 5.0, spends the night 4 ℃ of held.Take out back adjust pH to 5.5 next day, through the secondary ammonium sulfate precipitation method, for the first time according to adding ammonium sulfate solids 351g in the 1000ml solution; Add the ratio of 196g ammonium sulfate in every 1000ml the 2nd time; At 10 ℃ of following frozen centrifugations, rotating speed is 4000 rev/mins, and the time is 24 minutes.Abandoning supernatant, with protein precipitation with the dilution of 1: 4 vaal water and with the NaOH adjusting pH to 8.0 of 0.5mol/L.Adopt the G25 post to remove after the ammonium sulfate affinity column for preparing on the blended crude product solution.
1) collection screen is selected the egg of the hen production of good immune effect, and egg yolk is prepared ammoniumsulphate soln, removes after the ammonium sulfate, and bullion TK1 solution mixes goes up affinity column.
2) preparation affinity column
Select the antigenic type of affinity column: mixed bullion liquid derives from the anti-TK1-IgY antibody of the antigen immune of 23 peptides-KLH+20 peptide-KLH+28 peptide-KLH; Affinity column is prepared as 23 peptides+20 peptides+28 peptide mixing affinity columns, and the preparation affinity column requires to calculate corresponding antigen consumption according to selected 3 kinds of antibody blending ratios; I.e. 23 peptides; The per-cent of the amount of 20 peptides and 28 peptides is held 23 peptides with calculating good N, and C holds 20 peptides and C to hold the amount interlinkage of 28 peptides to receive affinity column.
3) antibody purification: under 2-8 ℃, will mix bullion liquid and add affinity column, 5 circulations, the speed when every ml affinity column is crossed post the 1st time is 0.2ml/min-0.25ml/min.The circulation back is with affinity column balanced solution flushing foreign protein, and through the repeated test more than 3 times, UV stops flushing all less than 0.010 o'clock; With the Actisep wash-out antibody of 1.5 times of affinity column volumes, wash-out is removed Actisep with the G25 post again, mixes collecting good antibody, surveys mixed UV value; In time transfer pH to 8.0, add protection reagent, preserve antibody in+4-8 degree refrigerator.
Instance 6: detect and identify TK1 test set installed reagents box
Under 100,000 grades clean environment, antibody is carried out packing.Like Fig. 2 (a), (b), (c) shown in, adopt the enhanced chemiluminescence dot blot that STK1 is carried out stdn and detect and identify, with easy, be up to the standards fast.
Embodiment 7: the sensitivity and the specificity of screening antibody
Identify the sensitivity and the specificity of antibody.1) Filter Sensitivity: Select the initial evaluation of qualified hens, ammonium sulfate solution was prepared with egg yolk, and then purified by affinity column prepared antibodies greater than 99% purity TK1 antibodies with enhanced luminescence immunoassay dot blot method, using standard TK1 Commodities (20,6.6,2.2 pM) standard curve has been recognized as a healthy selection of serum and cancer patients newly diagnosed untreated serum samples positive values, dot blot with enhanced chemiluminescence immunoassay to detect patients with cancer serum samples positive values should show varying degrees of positive values, healthy people should be below the detection threshold 2pM or immune response does not appear, do not react with human TK1 on healthy human serum with non-specific immunological cross-reactivity, or the blood of patients with tumor response breezy TK1 antibodies, antibodies for the unqualified, delete.
Identify the specificity of antibody
Cell cultures: adopt conventional cell culture processes, the CEM TK that experiment is used
+, CEM TK
-Cell uses the RPMI-1640 that contains 10% calf serum to cultivate cell count length to 20 * 10 in culturing bottle
6The time, with results cell with lysis buffer extract enchylema (lysis buffer: 10mM Tris-HCl, 250mM sucrose, 160mM KCl, 5.6mM NaF, 3.8mM MgCl2,5.0mM ATP, 0.2%NP-40, pH=7.5).Positive strain of the CEM TK1 of 20 micrograms/10 microlitres and the negative strain cell extract of CEM TK1 mix with the electrophoretic buffer of equivalent, natural gum electrophoresis (4-10% gradient), and test kit is provided by supplier Invitrogen company with operation.Carry out the western immunity printing and dyeing of TK1 antibody (half-dried electrophoretic transfer working method is provided by Bio-Rad).IgY TK1Ab (100,000X 1mg/ml) adopts human serum sample, IgY TK1Ab concentration (100,000X 1mg/ml).Like Fig. 3 (a) is the western printing and dyeing of TK antibody results, and CEM TK1 is positive, and strain has tangible electrophoresis band of TK1, and the negative strain of CEM TK1 does not manifest any band.Verified that antibody only has specific reaction with people TK1.
Fig. 3 (b) is that the western method of printing and dyeing is identified: sample source wherein is the lymphatic cancer patient before 1 example treatment, 1 example treatment is preceding/after head and neck cancer patient, 1 routine healthy subjects (no disease); Identify through some printing and dyeing/immunostimulant luminescent detection system (ECL); Be respectively 44,20,0.5 and 0.1pM.2 microlitre serum dilutions is that 10 microlitres mix with the electrophoretic buffer of equivalent, carries out western immune printing and dyeing of TK1 antibody after crossing natural gum electrophoresis (4-10% gradient).The result shows that the head and neck cancer patient before the treatment before lymphatic cancer patient and the treatment has a tangible electrophoresis band, and the head and neck cancer patient after the treatment and the serum T K1 of healthy subjects can observe hardly.Checking antibody and human serum do not have the non-specific immunity cross reaction.
Embodiment 8: identify the specificity and the sensitivity of TK1 antibody
Paraffin-embedded thick be 4 μ m section preparations, through the dewaxing aquation, the immersion target repair liquid of will cut into slices.According to the operation steps that supplier's (
System examination pigment agent box) provides, carry out histochemical stain.3%H is immersed in section
2O
2Solution, deactivation endogenous enzyme after 10 minutes was handled 30 minutes with closed reagent, added TK1 antibody, and at room temperature hatched 2 hours; Clean section with PBS solution; Add the EnVisiong mixture, and at room temperature hatched 40 minutes; Diaminobenzidine develops the color and with phenodin slide glass is carried out counterstaining.Positive with the negative human lymphoma cem cell of TK1 adopts the acetone fixed method, and dyeing thereafter and operation steps are ditto said.The TK1 that assesses each section expresses, and the microscopic field through＞10 is observed each tissue slice with the 200-400 magnification, calculates the percentage calculation of pressing the positive staining cell at last with the quantity that contains the TK1 positive cell in＞1000 cells.
Like Fig. 4, adopt a kind of lymph tumor cell strain: positive strain of CEM TK1 and the negative strain of CEM TK1 are that standard is identified.Collection of illustrative plates is presented in the tenuigenin in the positive strain cell of CEM TK1 and is positive staining (A) in various degree, but dyes in the negative strain of CEM TK1, and reaction (B) is negative.Malignant tumor patient tissue sample-endocervix cancer (C) and colorectal carcinoma (D).Collection of illustrative plates shows the dyeing characteristics: be positive staining in various degree in the tenuigenin in malignant tumour endocervix cancer and colon cancer cell, the part cell also dyes in the showed cell nuclear.Conclusion: TK1 antibody is to have specificity and sensitivity.
Embodiment 9: use TK1-IgY antibody
(1) detection method (being called 3 footworks) during the contracting of nitrocellulose film spot printing and dyeing/immunostimulant luminescent detection system (ECL)
Enhancing based on patent in 2002 is luminous-the immunity point method of printing and dyeing (ECL dot blot Assay).Set up method for quick.Its program is: get the biological fluid that early supplies examination person ante cibum, for example: 1) get fasting blood sample in early morning, centrifugal 4000 commentaries on classics/8-10 minutes, separation of serum.With the accurate point sample of the serum sample of 3 microlitres to nitrocellulose filter (HybandTMC, GE, UK) on, simultaneously, with different concns TK1 (20,6.6,2.2pM) do standard substance point sample (132).Air-dry 30 minutes is 7.5 TBS (20mmol/L Tris, 0.15mol/L NaCl) rinsing (2 minutes/2 times) with pH; 2) add 6% skimmed milk power with the configuration of TBS buffer solution, sealing is 1 hour under the room temperature, removes closed reagent; 3) add TK1 antibody, immune response 1.5 hours with the best diluted concentration of TBS configuration.After reaction is accomplished,, jolt washing 3 times (5 minutes/1 time) with the rapid rinsing of TBST (adding 0.1% polysorbas20 among the TBST) 2 times; 4) add biotinylation the 2nd antibody (by the optimum concn dilution), jolt reaction 40 minutes under the room temperature.After reaction is accomplished, the same method washing; 5) add avidin-horseradish peroxidase (streptavidin-Horse Radish Peroxidase).After the same method washing; Accurately reacted 1 minute with the ECL luminescence reagent, after the drying, this film phonograph seal is in bag film; Put into the CCD detecting instrument, the power of this luminous signal will be through CCD (Charged Coupled Device) imaging system analyser lock-on signal and scanning quantitative analysis.According to standard relatively, the concentration of TK1 is value-at-risk greater than normal healthy people level threshold value 2pM.Adopt this typical curve, calculate the change level of examinee TK1, corresponding to the rate of propagation of examinee's tumour cell.Adopt its accuracy of detection of antibody mediated immunity bonded detection method to reach below the 0.1pM.
Embodiment 10: the TK1-IgY antibody of the direct mark of applicating biotin
Like Fig. 5; Sulfonic group succinimide-direct mark the TK1-IgY of LC-vitamin H antibody (2143 test kits
Sulfo-NHS-LC-Biotinylation; Pierce, USA).The sulfonic group succinimide that takes out from-20 degree refrigerators-LC-vitamin H bottle; The method of pointing out is to specifications calculated; The vitamin H reagent of 20 times of mol ratios and the IgG antibody of 1 times of mole; The 2.2 milligrams of vitamin Hs of weighing are dissolved in ultrapure no any amine ionic water of 400 microlitres, and the vitamin H reagent of getting 24.1 microlitres joins among the PBS with 975.9 microlitres, dispose 2 milligrams of purifying TK1-IgY antibody.Ice bath was hatched two hours or was at room temperature reacted 30-60 minute.Adopt the molecule number of the vitamin H of HABA Assay certification mark, adopt desalting column to remove unnecessary vitamin H immediately.Adopt experimental example 5 methods, add biotinylation TK1 antibody (by the optimum concn dilution), jolt reaction 1.5 hours under the room temperature, after reaction is accomplished,, after the method washing, got into for the 5th step again according to experimental example 8 operations.The result is identical with 3 footworks, operates more simple and convenient.
Embodiment 11: identify the sensitivity of antibody
Specificity and sensitivity to the anti-people TK1-IgY antibody of multi-epitope antigen preparation combination are analyzed; The monospecific antibody of anti-people TK1-IgY antibody of multi-epitope antigen preparation combination (23 peptides+28 peptides+anti-people TK1-IgY antibody of 20 peptide multi-epitope antigens preparation combination was according to 1: 1: 1 ratio) and the immunity of 28 peptides, the monospecific antibody of the monospecific antibody of 23 peptides immunity and the immunity of 20 peptides compares.Be diagnosed as malignant tumor patient male serum to 11; Adopt enhancing electrochemiluminescent immunoassay dot blotting method to analyze; The result points out that multi-epitope antigen prepares combinatorial antibody and the immune single antibody of 28 peptides compares, in 50% serum specimen, and serum T K1 value rising 20%-50%.The monospecific antibody that multi-epitope antigen prepares combinatorial antibody and the immunity of 23 or 20 peptides compares, in 95% serum specimen, and serum T K1 value rising 20%-50%.Utilize the serum of 10 healthy subjects to analyze, multi-epitope antigen prepares the monospecific antibody of combinatorial antibody and the immunity of 28 peptides, and the monospecific antibody of the monospecific antibody of 23 peptides immunity and the immunity of 20 peptides compares, and does not have obvious variation.Explain that multi-epitope antigen prepares combinatorial antibody and improved sensitivity, better distinguish individual cells and whether be in the high or low of abnormality proliferation and increment degree thereof.Adopt ROC (experimenter's working curve, Analysis-It statistics computed in software) to confirm the raising of this antibody sensitivity like Fig. 6.According to the method described above; The examinee carries out the ROC mapping by data and preceding malignant tumour knurl patient 721 people of domestic routine clinical treatment that MEC provides the non-malignant tumors disease of participating in routine physical examination to amount to 4103 people, and the ROC mapping analysis is pointed out, is 2pM by setting risk threshold value; ROC TG-AUC value=0.96 (p＜0.0001); It is 236.49 that maximum likelihood method ratio (+) obviously raises, and specificity is 0.997, and sensitivity is 0.737; The anti-people TK1-IgY antibody of pointing out this specificity T K1 multi-epitope antigen preparation combination has highly sensitive and high specific, is applicable to the health check-up examination.
Instance 12: analyze the anti-TK1-IgY antibody of the relatively single epitope antigen preparation of specificity T K1 and sensitivity and the specificity that specificity T K1 multi-epitope antigen prepares the anti-TK1-IgY antibody of combination
The ROC analytical procedure is pointed out, the anti-TK1-IgY antibody of specificity T K1 multi-epitope antigen preparation combination, and maximum likelihood method ratio (+) obviously raises, and the meaningful rising of sensitivity is adopted the CHISQ methods analyst, p=0.028 (586/453,97/50).Form: sensitivity and the specificity of analyzing the IgY antibody and the combination IgY antibody that specificity T K1 multi-epitope antigen prepares of the relatively single epitope antigen preparation of specificity T K1
Embodiment 13: each peptide ratio range: 23 peptide 25-30%, and 20 peptide 20-25%, 28 peptide 50-55%, purpose is further to improve detection sensitivity and the antibody immunity from interference is used for early stage risk examination
Embodiment 14: the anti-TK1-IgY antibody that compares the preparation of commercial style mono-clonal TK1 antibody and Huarui Tongkang Biotechnology ltd
Adopt the method for quick of nitrocellulose film spot printing and dyeing/enhanced chemiluminescence detection system (ECL).
Its program is: getting early, supply examination person's biological fluid to detect ante cibum.According to standard relatively, the concentration of TK1 is value-at-risk greater than normal healthy people level threshold value 2pM.Adopt this typical curve, the convenient level of calculating examinee's TK1 in the treatment is changed, with relation corresponding to the rate of propagation of examinee's tumour cell.Adopt antibody mediated immunity bonded detection method can reach enough low accuracy of detection, particularly can detect the level difference of different healthy individuals TK1.The lowest detection amount can reach 0.1pM.Like Fig. 7 detected result: the serum that adopts the healthy no disease people of anti-TK1-IgY antibody test of Hua Ruitong health preparation; Its serum T K1 can not detect basically; But commercial style mono-clonal TK1 antibody (QED Bioscience Inc; San Diego, USA) serum with healthy subjects has tangible non-specific immunity cross reaction, and duplicate detection result is similar.Conclusion: (QED Bioscience Inc, San Diego USA) cannot be used for healthy population health check-up examination to commercial style mono-clonal TK1 antibody.
Embodiment 15: the serum-concentration according to healthy population distributes, and 95% crowd is under 1pM, and 5% at 1-2pM; We have set up serum T K1 (hereinafter to be referred as STK1) risk threshold value is 2pM; Normal people's group: STK1≤2pM, expression: cell normal growth or low propagation, the canceration risk is low; Risk people's group: STK1＞2pM, expression cellular abnormality propagation raises, and process is the risk rising of cancer disease in the several years.Risk group and Chinese's cancer occurrence frequency ratio, the tracking results in 6 years shows: normal group and rising group crowd relatively, process is elevated to 30 times for the cancer disease risks, all data are passed through statistical analysis, p＜0.005 has statistical significance.
Rising is organized the tracking that 170 people and normal cohort 6,354 people of TK1 have carried out 5-72 month, conclusion to TK1: TK1 rising group process is that the risk of malignant tumour raises 30 times.
According to examinee's STK1 level, set up the basal level of the individual TK1 of healthy person, for example there is not the healthy examinee of tumor disease; Need get the sample in 1st month for the first time, analyze the level of STK1,3 middle of the month; Carry out duplicate detection 3 times,, draw the basal level of healthy individuals TK1 3 times detected result calculating mean value; The healthy examinee of no tumor disease generally is lower than 1pM, and during the annual later on routine physical examination, the STK1 value of utilization detection compares with the basal level of individual TK1.
To maybe process being that the examinee's of cancer disease risks prediction is a STK1 value of utilizing the basic value of individual STK1 and annual test later on; STK1 level to both compares, and comprising: Individual Age, and examine the basis of STK1 value and individual former STK1 now and compare; If should confirm to have the preceding disease of cancer/innocent tumour disease/chronic disease relevant by individuality with tumor disease; But the level of STK1 still is lower than risk threshold value, and it is low that the examinee is evaluated as cellular abnormality propagation level, and process is that the risk of cancer disease is low; If should confirm to have the preceding disease of cancer/innocent tumour disease/chronic disease relevant with tumor disease by individuality, the level rising 1-2 of STK1 doubly surpasses 2pM, and the examinee is evaluated as cellular abnormality propagation level and raises, and the early warning process is that the risk of cancer disease raises.
Claims (8)
1. a high specific, highly sensitive anti-people TK1 antibody; It is characterized in that the TK1 albumen that said antibody is selected comprises: TK1 protein; Have enzymic activity/inactive monomer, 2 or 4 aggressiveness TK1 protein, or the albumen composition or the inhibition of TK1 protein and the formation of other molecules.
2. epitope, said decision epi-position comprises that human cervical carcinoma cell TK1 monomer N holds 23 peptides, C to hold 20 peptides and C to hold 28 peptides, its aminoacid sequence respectively as follows: N holds 23 peptides (3-25): CINLPTVLPGSPSKTRGQIQVIL; C holds 20 peptides (206-225): CPVPGKPGEAVAARKLFAPQ and C hold 28 peptides (198-225): AGPDNKENCPVPGKPGEAVAARKLFAPQ.
3. method of utilizing the described antigenic determinant of claim 2 to prepare antibody; It is characterized in that said method comprises that to utilize claim 2 described three sections polypeptide immune sequences crosslinked with KLH respectively; Immune hen extracts yolk liquid respectively, adopts water extraction preparation antibody-solutions; With the affinity column antibody purification of described antigenic determinant preparation, just sift out high specific, highly sensitive antibody respectively.
4. high specific, the anti-people TK1-IgY of the highly sensitive multi-epitope antibody of a combinatorial antibody preparation that utilizes the described method gained of claim 3; It is characterized in that described antibody comprises by mass percentage: the monospecific antibody of said 23 peptides immunity is 25%～45%; The monospecific antibody of said 20 peptides immunity is that the monospecific antibody of 20%～40%, 28 peptides immunity is 20%～55%.
5. high specific, the anti-people TK1-IgY of the highly sensitive multi-epitope antibody of a combinatorial antibody preparation that utilizes the described method gained of claim 3; It is characterized in that described antibody comprises by mass percentage: the monospecific antibody of said 23 peptides immunity is 25%～30%; The monospecific antibody of said 20 peptides immunity is that the monospecific antibody of 20%～25%, 28 peptides immunity is 50%～55%.
6. one kind with the described antibody of arbitrary claim in the claim 3 to 5; Its characteristic comprises at the TK1 albumen with described antibody selection: TK1 protein; Have enzymic activity/inactive monomer, 2 or 4 aggressiveness TK1 protein, or the albumen composition or the inhibition of TK1 protein and the formation of other molecules.
7. anti-people TK1-IgY detection of antibodies method to the coordination described in the claim 4 combination: detection method, the direct mark vitamin H of antibody method when said detection method comprises contracting of nitrocellulose film spot printing and dyeing/immunostimulant luminescent detection system (ECL).
8. one kind is utilized the anti-people TK1-IgY of the described combination of arbitrary claim antibody in the claim 4 to 5 to combine with chemiluminescence detection system to be used for that crowd's health check-up examination infantile tumour detects and the test kit of Risk-warning.
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